Cell and molecular biololgy, concepts and experiments 6th ed g karp (wiley, 2010) 1

JWCL151_fendpapers.qxd 9/14/09 11:09 AM Page Nobel Prizes Awarded for Research in Cell and Molecular Biology Since 1958 Year Recipient* Prize Area of Research Pages in Text 2008 Francoise Barré-Sinoussi Luc Montagnier Harald zur Hausen Martin Chalfie Osamu Shimomura Roger Tsien Mario R Capecchi Martin J Evans Oliver Smithies Andrew Z Fire Craig C Mello Roger D Kornberg Richard Axel Linda B Buck Aaron Ciechanover Avram Hershko Irwin Rose Peter Agre Roderick MacKinnon Sydney Brenner John Sulston H Robert Horvitz John B Fenn Koichi Tanaka Kurt Wüthrich Leland H Hartwell Tim Hunt Paul Nurse Arvid Carlsson Paul Greengard Eric Kandel Günter Blobel Robert Furchgott Louis Ignarro Ferid Murad Jens C Skou Paul Boyer John Walker Stanley B Prusiner Rolf M Zinkernagel Peter C Doherty Edward B Lewis Christiane Nüsslein-Volhard Eric Wieschaus Alfred Gilman Martin Rodbell Kary Mullis Michael Smith Richard J Roberts Phillip A Sharp M & P** Discovery of HIV 23 Chemistry Role of HPV in cancer Discovery and development of GFP 654 267, 720 M&P Development of techniques for knockout mice 760 M&P RNA Interference 449, 762 Chemistry M&P Transcription in eukaryotes Olfactory receptors 427, 481 622 Chemistry Ubiquitin and proteasomes 529 Chemistry Structure of membrane channels Introduction of C elegans as a model organism Apoptosis in C elegans Electrospray ionization in MS MALDI in MS NMR analysis of proteins Control of the cell cycle 2007 2006 2004 2003 2002 2001 2000 1999 1998 1997 1996 1995 1994 1993 M&P Chemistry M&P 146, 148 17 643 740 740 56 564, 600 M&P Synaptic transmission and signal transduction 163 605 M&P M&P Protein trafficking NO as intercellular messenger 276 641 Chemistry Naϩ/Kϩ-ATPase Mechanism of ATP synthesis 153 195 M&P M&P Protein nature of prions Recognition of virus-infected cells by the immune system Genetic control of embryonic development 64 709 M&P M&P Chemistry M&P Structure and function of GTP-binding (G) proteins Polymerase chain reaction (PCR) Site-directed mutagenesis (SDM) Intervening sequences EP12 610 751 760 438 JWCL151_fendpapers.qxd 9/14/09 11:09 AM Page Year Recipient* Prize Area of Research 1992 Edmond Fischer Edwin Krebs Erwin Neher Bert Sakmann J Michael Bishop Harold Varmus Thomas R Cech Sidney Altman Johann Deisenhofer Robert Huber Hartmut Michel Susumu Tonegawa M&P Alteration of enzyme activity by phosphorylation/dephosphorylation Measurement of ion flux by patch-clamp recording Cellular genes capable of causing malignant transformation Ability of RNA to catalyze reactions 1991 1989 1988 1987 1986 1985 1984 1983 1982 1980 1978 1976 1975 1974 1972 Rita Levi-Montalcini Stanley Cohen Michael S Brown Joseph L Goldstein Georges Köhler Cesar Milstein Niels K Jerne Bruce Merrifield Barbara McClintock Aaron Klug Paul Berg Walter Gilbert Frederick Sanger Baruj Bennacerraf Jean Dausset George D Snell Werner Arber Daniel Nathans Hamilton O Smith Peter Mitchell D Carleton Gajdusek David Baltimore Renato Dulbecco Howard M Temin Albert Claude Christian de Duve George E Palade Gerald Edelman Rodney R Porter Christian B Anfinsen M&P M&P Chemistry 112, 614 147 677 469 Chemistry Bacterial photosynthetic reaction center 213 M&P DNA rearrangements responsible for antibody diversity Factors that affect nerve outgrowth 696 M&P M&P 372 Regulation of cholesterol metabolism and endocytosis Monoclonal antibodies 312 Antibody formation Chemical synthesis of peptides Mobile elements in the genome Structure of nucleic acid-protein complexes Recombinant DNA technology DNA sequencing technology 687 746 402 76 M&P Major histocompatibility complex 699 M&P Restriction endonuclease technology 746 Chemistry Chemiosmotic mechanism of oxidative phosphorylation Prion-based diseases Reverse transcriptase and tumor virus activity 181 M&P Structure and function of internal components of cells 267 M&P Immunoglobulin structure 693 Chemistry Relationship between primary and tertiary structure of proteins Mechanism of hormone action and cyclic AMP Nerve impulse propagation and transmission Role of sugar nucleotides in carbohydrate synthesis Genetic structure of viruses M&P Chemistry M&P Chemistry Chemistry M&P M&P 1971 Earl W Sutherland M&P 1970 Bernard Katz Ulf von Euler Luis F Leloir M&P Max Delbrück Alfred D Hershey Salvador E Luria M&P 1969 Pages in Text Chemistry 763 748 753 64 676 62 614 160 280 22, 415 JWCL151_fendpapers.qxd 9/14/09 11:09 AM Page Year Recipient* Prize Area of Research 1968 H Gobind Khorana Marshall W Nirenberg Robert W Holley Peyton Rous Francois Jacob Andre M Lwoff Jacques L Monod Dorothy C Hodgkin John C Eccles Alan L Hodgkin Andrew F Huxley Francis H C Crick James D Watson Maurice H F Wilkins John C Kendrew Max F Perutz Melvin Calvin M&P Genetic code M&P M&P Transfer RNA structure Tumor viruses Bacterial operons and messenger RNA F MacFarlane Burnet Peter B Medawar Arthur Kornberg Severo Ochoa George W Beadle Joshua Lederberg Edward L Tatum Frederick Sanger M&P 1966 1965 1964 1963 1962 1961 1960 1959 1958 Pages in Text 456 457 676 500, 421 Chemistry M&P X-ray structure of complex biological molecules Ionic basis of nerve membrane potentials 740 159 M&P Three-dimensional structure of DNA 386 Chemistry M&P Three-dimensional structure of globular proteins Biochemistry of CO2 assimilation during photosynthesis Clonal selection theory of antibody formation Synthesis of DNA and RNA M&P Gene expression Chemistry Primary structure of proteins Chemistry *In a few cases, corecipients whose research was in an area outside of cell and molecular biology have been omitted from this list **Medicine and Physiology 56 221 687 538, 456 420 54 JWCL151_fm_i-xviii.qxd 8/7/09 8:54 PM Page i This online teaching and learning environment integrates the entire digital textbook with the most effective instructor and student resources to fit every learning style With WileyPLUS: • Students achieve concept mastery in a rich, structured environment that’s available 24/7 • Instructors personalize and manage 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JWCL151_fm_i-xviii.qxd 8/7/09 8:54 PM Page iii th edition Cell and Molecular Biology Concepts and Experiments Gerald Karp John Wiley & Sons, Inc JWCL151_fm_i-xviii.qxd 8/7/09 8:54 PM Page iv ACQUISITIONS EDITOR PROJECT EDITOR SR PRODUCTION EDITOR MARKETING MANAGER SENIOR PHOTO EDITOR SENIOR DESIGNER ILLUSTRATION EDITOR ILLUSTRATIONS MEDIA EDITOR COVER PHOTO Kevin Witt Merilatt Staat Patricia McFadden Ashaki Charles Hilary Newman Madelyn Lesure Anna Melhorn Imagineering, Inc Linda Muriello From Shigeo Takamori et al., courtesy of Reinhard Jahn of the Max-Planck Institute for Biophysical Chemistry, Cell 127:841, 2006 PRODUCTION SERVICES Furino Production This book was set in 10.5/12 Adobe Caslon by Aptara, and printed and bound by RR Donnelley The cover was printed by RR Donnelley This book is printed on acid free paper Copyright © 2010 John Wiley & Sons, Inc All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, scanning or otherwise, except as permitted under Sections 107 or 108 of the 1976 United States Copyright Act, without either the prior written permission of the Publisher, or authorization through payment of the appropriate per-copy fee to the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, website www.copyright.com Requests to the Publisher for permission should be addressed to the Permissions Department, John Wiley & Sons, Inc., 111 River Street, Hoboken, NJ 07030-5774, (201)748-6011, fax (201)748-6008, website http://www.wiley.com/go/permissions To order books or for customer service, please call 1-800-CALL WILEY (225-5945) ISBN-13 978-0-470-48337-4 Printed in the United States of America 10 JWCL151_fm_i-xviii.qxd 8/7/09 8:54 PM Page v About the Author G erald C Karp received a bachelor’s degree from UCLA and a Ph.D from the University of Washington He conducted postdoctoral research at the University of Colorado Medical Center before joining the faculty at the University of Florida Gerry is the author of numerous research articles on the cell and molecular biology of early development His interests have included the synthesis of RNA in early embryos, the movement of mesenchyme cells during gastrulation, and cell determination in slime molds For 13 years, he taught courses in molecular, cellular, and developmental biology at the University of Florida During this period, Gerry coauthored a text in developmental biology with N John Berrill and authored a text in cell and molecular biology Finding it impossible to carry on life as both full-time professor and author, Gerry gave up his faculty position to concentrate on writing He hopes to revise this text every three years About the Cover A molecular model of the membrane of a synaptic vesicle Within nerve cells, a synaptic vesicle consists of a cellular membrane surrounding a soluble compartment filled with neurotransmitter molecules Vesicles of this type are assembled in the vicinity of a nerve cell’s nucleus and then transported to the tip of the axon There the vesicle awaits the arrival of a nerve impulse that will induce it to fuse with the overlying plasma membrane, releasing its contents into the narrow cleft that separates the nerve cell from a neighboring cell The threedimensional model of this membrane was constructed using known structures of the various proteins along with information on their relative numbers obtained from the analysis of purified synaptic vesicles The image on the front cover shows a synaptic vesicle that has been cut in half; the lipid bilayer that forms the core of the vesicle membrane is shown in green The image on the back cover shows the surface structure of an intact vesicle Most of the proteins present in this membrane are required for the interaction of the vesicle with the plasma membrane The large blue protein at the lower right of the vesicle contains a ring of subunits that rotates within the lipid bilayer as the protein pumps hydrogen ions into the vesicle The elevated concentration of hydrogen ions within the vesicle is subsequently used as an energy source for the uptake of neurotransmitter molecules from the surrounding cytosol These images provide the most comprehensive model of any cellular membrane yet to be studied and they reveal how much this membrane is dominated by protein—both within the bilayer itself and on both membrane surfaces (From Shigeo Takamori et al., courtesy of Reinhard Jahn of the Max-Planck Institute for Biophysical Chemistry, Cell 127:841, 2006.) JWCL151_fm_i-xviii.qxd 8/7/09 8:54 PM Page vi To Patsy and Jenny JWCL151_ch02_031-083.qxd 44 6/10/09 1:44 PM Page 44 Chapter THE CHEMICAL BASIS OF LIFE CH2OH CH2OH H O OH H OH H HO H H H C C H OH HO OH CH2OH OH H H C H O 3C H β-D-Glucopyranose 2C glucose undergoes self-reaction to form a pyranose ring (i.e., a sixmembered ring), two stereoisomers are generated The two isomers are in equilibrium with each other through the open-chain form of the Linking Sugars Together Sugars can be joined to one another by covalent glycosidic bonds to form larger molecules Glycosidic bonds form by reaction between carbon atom C1 of one sugar and the hydroxyl group of another sugar, generating a —C—O—C— linkage between the two sugars As discussed below (and indicated in Figures 2.16 and 2.17), sugars can be joined by quite a variety of different glycosidic bonds Molecules composed of only two sugar units are disaccharides (Figure 2.16) Disaccharides serve primarily as readily available energy stores Sucrose, or table sugar, is a major component of plant sap, which carries chemical energy from one part of the plant to another Lactose, present in the milk of most mammals, supplies newborn mammals with fuel for early growth and development Lactose in the diet is hy- H H OH HO H HOCH2 O H H (α) 2 H O H O HO OH OH CH2OH H (a) Lactose 6 5 CH2OH HO H H OH H CH2OH H O H (β) OH H O H OH H OH H OH OH molecule By convention, the molecule is an ␣-pyranose when the OH group of the first carbon projects below the plane of the ring, and a ␤-pyranose when the hydroxyl group projects upward drolyzed by the enzyme lactase, which is present in the plasma membranes of the cells that line the intestine Many people lose this enzyme after childhood and find that eating dairy products causes digestive discomfort Sugars may also be linked together to form small chains called oligosaccharides (oligo ϭ few) Most often such chains are found covalently attached to lipids and proteins, converting them into glycolipids and glycoproteins, respectively Oligosaccharides are particularly important on the glycolipids and glycoproteins of the plasma membrane, where they project from the cell surface (see Figure 4.4c) Because oligosaccharides may be composed of many different combinations of sugar units, these carbohydrates can play an informational role; that is, they can serve to distinguish one type of cell from another and help mediate specific interactions of a cell with its surroundings By the middle of the nineteenth century, it was known that the blood of people suffering from diabetes had a sweet taste due to an elevated level of glucose, the key sugar in energy metabolism Claude Bernard, a prominent French physiologist of the period, was looking for the cause of diabetes by investigating the source of blood sugar It was assumed at the time that any sugar present in a human or an animal had to have been previously consumed in the diet Working with dogs, Bernard found that, even if the animals were placed on a diet totally lacking carbohydrates, their blood still contained a normal amount of glucose Clearly, glucose could be formed in the body from other types of compounds After further investigation, Bernard found that glucose enters the blood from the liver Liver tissue, he found, contains an insoluble polymer of glucose he named glycogen Bernard concluded that various food materials (such as proteins) were carried to the liver where they were chemically converted to glucose and stored as glycogen Then, as the body needed sugar for fuel, the glycogen in the liver was transformed to glucose, which was released into the bloodstream to satisfy glucose-depleted tissues In Bernard’s hypothesis, the balance between glycogen formation and glycogen breakdown in the liver was the prime determinant in maintaining the relatively constant (homeostatic) level of glucose in the blood Polysaccharides Sucrose CH2OH H HO α-D-Glucopyranose FIGURE 2.15 Formation of an ␣- and ␤-pyranose When a molecule of O H H OH O OH H H OH (b) FIGURE 2.16 Disaccharides Sucrose and lactose are two of the most common disaccharides Sucrose is composed of glucose and fructose joined by an ␣(1 S 2) linkage, whereas lactose is composed of glucose and galactose joined by a ␤(1 S 4) linkage JWCL151_ch02_031-083.qxd 6/10/09 1:44 PM Page 45 2.5 FOUR TYPES OF BIOLOGICAL MOLECULES 45 Glycogen (a) 2 (b) Starch (c) Cellulose FIGURE 2.17 Three polysaccharides with identical sugar monomers but dramatically different properties Glycogen (a), starch (b), and cellulose (c) are each composed entirely of glucose subunits, yet their chemical and physical properties are very different due to the distinct ways that the monomers are linked together (three different types of linkages are indicated by the circled numbers) Glycogen molecules are the most highly branched, starch molecules assume a helical arrangement, and cellulose molecules are unbranched and highly extended Whereas glycogen and starch are energy stores, cellulose molecules are bundled together into tough fibers that are suited for their structural role Colorized electron micrographs show glycogen granules in a liver cell, starch grains (amyloplasts) in a plant seed, and cellulose fibers in a plant cell wall; each is indicated by an arrow [PHOTO INSETS: (TOP) Bernard’s hypothesis proved to be correct The molecule he named glycogen is a type of polysaccharide—a polymer of sugar units joined by glycosidic bonds Glycogen serves as a storehouse of surplus chemical energy in most animals Human skeletal muscles, for example, typically contain enough glycogen to fuel about 30 minutes of moderate activity Depending on various factors, glycogen typically ranges in molecular weight from about one to four million daltons When stored in cells, glycogen is highly concentrated in what appears as dark-staining, irregular granules in electron micrographs (Figure 2.17a, right) Most plants bank their surplus chemical energy in the form of starch, which like glycogen is also a polymer of glucose Potatoes and cereals, for example, consist primarily of starch Starch is actually a mixture of two different polymers, amylose and amylopectin Amylose is an unbranched, helical Glycogen and Starch: Nutritional Polysaccharides Glycogen is a branched polymer containing only one type of monomer: glucose (Figure 2.17a) Most of the sugar units of a glycogen molecule are joined to one another by ␣(1 S 4) glycosidic bonds (type bond in Figure 2.17a) Branch points contain a sugar joined to three neighboring units rather than to two, as in the unbranched segments of the polymer The extra neighbor, which forms the branch, is linked by an ␣(1 S 6) glycosidic bond (type bond in Figure 2.17a) DON FAWCETT/VISUALS UNLIMITED; (CENTER) JEREMY BURGESS/PHOTO RESEARCHERS; (BOTTOM) CABISCO/VISUALS UNLIMITED.] JWCL151_ch02_031-083.qxd 46 6/10/09 1:44 PM Page 46 Chapter THE CHEMICAL BASIS OF LIFE molecule whose sugars are joined by ␣(1 S 4) linkages (Figure 2.17b), whereas amylopectin is branched Amylopectin differs from glycogen in being much less branched and having an irregular branching pattern Starch is stored as densely packed granules, or starch grains, which are enclosed in membranebound organelles (plastids) within the plant cell (Figure 2.17b, right) Although animals don’t synthesize starch, they possess an enzyme (amylase) that readily hydrolyzes it Cellulose, Chitin, and Glycosaminoglycans: Structural Polysaccharides Whereas some polysaccharides constitute easily digested energy stores, others form tough, durable structural materials Cotton and linen, for example, consist largely of cellulose, which is the major component of plant cell walls Cotton textiles owe their durability to the long, unbranched cellulose molecules, which are ordered into side-by-side aggregates to form molecular cables (photo, Figure 2.17c) that are ideally constructed to resist pulling (tensile) forces Like glycogen and starch, cellulose consists solely of glucose monomers; its properties differ dramatically from these other polysaccharides because the glucose units are joined by ␤(1 S 4) linkages (bond in Figure 2.17c) rather than ␣(1 S 4) linkages Ironically, multicellular animals (with rare exception) lack the enzyme needed to degrade cellulose, which happens to be the most abundant organic material on Earth and rich in chemical energy Animals that “make a living” by digesting cellulose, such as termites and sheep, so by harboring bacteria and protozoa that synthesize the necessary enzyme, cellulase Not all biological polysaccharides consist of glucose monomers Chitin is an unbranched polymer of the sugar N-acetylglucosamine, which is similar in structure to glucose but has an acetyl amino group instead of a hydroxyl group bonded to the second carbon atom of the ring CH2OH H O H H OH HO H OH H HNCOCH3 N -Acetylglucosamine Chitin occurs widely as a structural material among invertebrates, particularly in the outer covering of insects, spiders, and crustaceans Chitin is a tough, resilient, yet flexible material not unlike certain plastics Insects owe much of their success to this highly adaptive polysaccharide (Figure 2.18) Another group of polysaccharides that has a more complex structure is the glycosaminoglycans (or GAGs) Unlike other polysaccharides, they have the structure —A—B—A—B—, where A and B represent two different sugars The best-studied GAG is heparin, which is secreted by cells in the lungs and other tissues in response to tissue injury Heparin inhibits blood coagulation, thereby preventing the formation of clots that can block the flow of blood to the heart or lungs Heparin accomplishes this feat by activating an inhibitor (antithrombin) of a key enzyme (thrombin) that is FIGURE 2.18 Chitin is the primary component of the glistening outer skeleton of this grasshopper (FROM ROBERT AND LINDA MITCHELL.) required for blood coagulation Heparin, which is normally extracted from pig tissue, has been used for decades to prevent blood clots in patients following major surgery Unlike heparin, most GAGs are found in the spaces that surround cells, and their structure and function are discussed in Section 7.1 The most complex polysaccharides are found in plant cell walls (Section 7.6) Lipids Lipids are a diverse group of nonpolar biological molecules whose common properties are their ability to dissolve in organic solvents, such as chloroform or benzene, and their inability to dissolve in water—a property that explains many of their varied biological functions Lipids of importance in cellular function include fats, steroids, and phospholipids Fats Fats consist of a glycerol molecule linked by ester bonds to three fatty acids; the composite molecule is termed a triacylglycerol (Figure 2.19a) We will begin by considering the structure of fatty acids Fatty acids are long, unbranched hydrocarbon chains with a single carboxyl group at one end (Figure 2.19b) Because the two ends of a fatty acid molecule have a very different structure, they also have different properties The hydrocarbon chain is hydrophobic, whereas the carboxyl group (—COOH), which bears a negative JWCL151_ch02_031-083.qxd 6/10/09 1:44 PM Page 47 2.5 FOUR TYPES OF BIOLOGICAL MOLECULES Glycerol moiety CH2 Fatty acid tail O 47 Water C O O CH C O O CH2 C O (a) Stearic acid HO O H H H H H H H H H H H H H H H H H C C C C C C C C C C C C C C C C C C H H H H H H H H H H H H H H H H H H (b) Tristearate FIGURE 2.20 Soaps consist of fatty acids In this schematic drawing of a soap micelle, the nonpolar tails of the fatty acids are directed inward, where they interact with the greasy matter to be dissolved The negatively charged heads are located at the surface of the micelle, where they interact with the surrounding water Membrane proteins, which also tend to be insoluble in water, can also be solubilized in this way by extraction of membranes with detergents (c) Linseed oil (d) FIGURE 2.19 Fats and fatty acids (a) The basic structure of a triacylglycerol (also called a triglyceride or a neutral fat) The glycerol moiety, indicated in orange, is linked by three ester bonds to the carboxyl groups of three fatty acids whose tails are indicated in green (b) Stearic acid, an 18-carbon saturated fatty acid that is common in animal fats (c) Spacefilling model of tristearate, a triacylglycerol containing three identical stearic acid chains (d ) Space-filling model of linseed oil, a triacylglycerol derived from flax seeds that contains three unsaturated fatty acids (linoleic, oleic, and linolenic acids) The sites of unsaturation, which produce kinks in the molecule, are indicated by the yellow-orange bars which consists of fatty acids In past centuries, soaps were made by heating animal fat in strong alkali (NaOH or KOH) to break the bonds between the fatty acids and the glycerol Today, most soaps are made synthetically Soaps owe their grease-dissolving capability to the fact that the hydrophobic end of each fatty acid can embed itself in the grease, whereas the hydrophilic end can interact with the surrounding water As a result, greasy materials are converted into complexes (micelles) that can be dispersed by water (Figure 2.20) Fatty acids differ from one another in the length of their hydrocarbon chain and the presence or absence of double bonds Fatty acids present in cells typically vary in length from 14 to 20 carbons Fatty acids that lack double bonds, such as stearic acid (Figure 2.19b), are described as saturated; those possessing double bonds are unsaturated Naturally occurring fatty acids have double bonds in the cis configuration Double bonds (of the cis configuration) H C C C H C as opposed to C cis charge at physiological pH, is hydrophilic Molecules having both hydrophobic and hydrophilic regions are said to be amphipathic; such molecules have unusual and biologically important properties The properties of fatty acids can be appreciated by considering the use of a familiar product: soap, C H C H C trans produce kinks in a fatty acid chain Consequently, the more double bonds that fatty acid chains possess, the less effectively these long chains can be packed together This lowers the temperature at which a fatty acid-containing lipid melts Tristearate, whose fatty acids lack double bonds (Figure 2.19c), is a common component of animal fats and remains in a solid JWCL151_ch02_031-083.qxd 48 6/10/09 1:44 PM Page 48 Chapter THE CHEMICAL BASIS OF LIFE state well above room temperature In contrast, the profusion of double bonds in vegetable fats accounts for their liquid state—both in the plant cell and on the grocery shelf—and for their being labeled as “polyunsaturated.” Fats that are liquid at room temperature are described as oils Figure 2.19d shows the structure of linseed oil, a highly volatile lipid extracted from flax seeds, that remains a liquid at a much lower temperature than does tristearate Solid shortenings, such as margarine, are formed from unsaturated vegetable oils by chemically reducing the double bonds with hydrogen atoms (a process termed hydrogenation) The hydrogenation process also converts some of the cis double bonds into trans double bonds, which are straight rather than kinked This process generates partially hydrogenated or trans-fats A molecule of fat can contain three identical fatty acids (as in Figure 2.19c), or it can be a mixed fat, containing more than one fatty acid species (as in Figure 2.19d ) Most natural fats, such as olive oil or butterfat, are mixtures of molecules having different fatty acid species Fats are very rich in chemical energy; a gram of fat contains over twice the energy content of a gram of carbohydrate (for reasons discussed in Section 3.1) Carbohydrates function primarily as a short-term, rapidly available energy source, whereas fat reserves store energy on a long-term basis It is estimated that a person of average size contains about 0.5 kilograms (kg) of carbohydrate, primarily in the form of glycogen This amount of carbohydrate provides approximately 2000 kcal of total energy During the course of a strenuous day’s exercise, a person can virtually deplete his or her body’s entire store of carbohydrate In contrast, the average person contains approximately 16 kg of fat (equivalent to 144,000 kcal of energy), and as we all know, it can take a very long time to deplete our store of this material Because they lack polar groups, fats are extremely insoluble in water and are stored in cells in the form of dry lipid droplets Since lipid droplets not contain water as glycogen granules, they represent an extremely concentrated storage fuel In many animals, fats are stored in special cells (adipocytes) whose cytoplasm is filled with one or a few large lipid droplets Adipocytes exhibit a remarkable ability to change their volume to accommodate varying quantities of fat Steroids are built around a characteristic four-ringed hydrocarbon skeleton One of the most important steroids is cholesterol, a component of animal cell membranes and a precursor for the synthesis of a number of steroid hormones, such as testosterone, progesterone, and estrogen (Figure 2.21) Cholesterol is largely absent from plant cells, which is why vegetable oils are considered “cholesterol-free,” but plant cells may contain large quantities of related compounds Steroids The chemical structure of a common phospholipid is shown in Figure 2.22 The molecule resembles a fat (triacylglycerol), but has only two fatty acid chains rather than three; it is a diacylglycerol The third hydroxyl of the glycerol backbone is covalently bonded to a phosphate group, which in turn is covalently bonded to a small polar group, such Phospholipids CH3 CH3 CH3 Cholesterol CH3 CH3 HO OH CH3 Testosterone CH3 O OH CH3 Estrogen HO FIGURE 2.21 The structure of steroids All steroids share the basic four-ring skeleton The seemingly minor differences in chemical structure between cholesterol, testosterone, and estrogen generate profound biological differences as choline, as shown in Figure 2.22 Thus, unlike fat molecules, phospholipids contain two ends that have very different properties: the end containing the phosphate group has a distinctly hydrophilic character; the other end composed of the two fatty acid tails has a distinctly hydrophobic character Because phospholipids function primarily in cell membranes, Phosphate CH3 – O + H3C N CH2 CH2 O P O CH2 CH3 Choline O O H H H H H H H H H H H H H H H H H HC O C C C C C C C C C C C C C C C C C C H H H H H H H H H H H H H H H H H H O H H H H H H H H H H H H H H H H H H2C O C C C C C C C C C C C C C C C C C C H H H H H H H H H H H H H H H H H H Polar head group Glycerol backbone Fatty acid chains FIGURE 2.22 The phospholipid phosphatidylcholine The molecule consists of a glycerol backbone whose hydroxyl groups are covalently bonded to two fatty acids and a phosphate group The negatively charged phosphate is also bonded to a small, positively charged choline group The end of the molecule that contains the phosphorylcholine is hydrophilic, whereas the opposite end, consisting of the fatty acid tail, is hydrophobic The structure and function of phosphatidylcholine and other phospholipids are discussed at length in Section 4.3 JWCL151_ch02_031-083.qxd 6/10/09 1:44 PM Page 49 2.5 FOUR TYPES OF BIOLOGICAL MOLECULES and because the properties of cell membranes depend on their phospholipid components, they will be discussed further in Sections 4.3 and 15.2 in connection with cell membranes Proteins Proteins are the macromolecules that carry out virtually all of a cell’s activities; they are the molecular tools and machines that make things happen As enzymes, proteins vastly accelerate the rate of metabolic reactions; as structural cables, proteins provide mechanical support both within cells and outside their perimeters (Figure 2.23a); as hormones, growth factors, and gene activators, proteins perform a wide variety of regulatory functions; as membrane receptors and transporters, proteins determine what a cell reacts to and what types of substances enter or leave the cell; as contractile filaments and molecular motors, proteins constitute the machinery for biological movements Among their many other functions, proteins act as antibodies, serve as toxins, form blood clots, absorb or refract light (Figure 2.23b), and transport substances from one part of the body to another How can one type of molecule have so many varied functions? The explanation resides in the virtually unlimited molecular structures that proteins, as a group, can assume Each protein, however, has a unique and defined structure that enables it to carry out a particular function Most importantly, proteins have shapes and surfaces that allow them to interact selectively with other molecules Proteins, in other words, exhibit a high degree of specificity It is possible, for example, for a particular DNA-cutting enzyme to recognize a segment of DNA containing one specific sequence of eight (a) FIGURE 2.23 Two examples of the thousands of biological structures composed predominantly of protein These include (a) feathers, which are adaptations in birds for thermal insulation, flight, and sex recogni- 49 nucleotides, while ignoring all the other 65,535 possible sequences composed of this number of nucleotides The Building Blocks of Proteins Proteins are polymers made of amino acid monomers Each protein has a unique sequence of amino acids that gives the molecule its unique properties Many of the capabilities of a protein can be understood by examining the chemical properties of its constituent amino acids Twenty different amino acids are commonly used in the construction of proteins, whether from a virus or a human There are two aspects of amino acid structure to consider: that which is common to all of them and that which is unique to each We will begin with the shared properties The Structures of Amino Acids All amino acids have a carboxyl group and an amino group, which are separated from each other by a single carbon atom, the ␣-carbon (Figure 2.24a,b) In a neutral aqueous solution, the ␣-carboxyl group loses its proton and exists in a negatively charged state (—COOϪ), and the ␣-amino group accepts a proton and exists in a positively charged state (NHϩ ) (Figure 2.24b) We saw on page 43 that carbon atoms bonded to four different groups can exist in two configurations (stereoisomers) that cannot be superimposed on one another Amino acids also have asymmetric carbon atoms With the exception of glycine, the ␣-carbon of amino acids bonds to four different groups so that each amino acid can exist in either a D or an L form (Figure 2.25) Amino acids used in the synthesis of a protein on a ribosome are always L-amino acids The “selection” of L-amino acids must have occurred very early in cellular evolution and has been conserved for billions of years Microorgan- (b) tion; and (b) the lenses of eyes, as in this net-casting spider, which focus light rays (A: DARRELL GULIN/GETTY IMAGES; B: MANTIS WILDLIFE FILMS/OXFORD SCIENTIFIC FILMS/ANIMALS ANIMALS.) JWCL151_ch02_031-083.qxd 50 6/10/09 1:44 PM Page 50 Chapter THE CHEMICAL BASIS OF LIFE isms, however, use D-amino acids in the synthesis of certain small peptides, including those of the cell wall and several antibiotics (e.g., gramicidin A) During the process of protein synthesis, each amino acid becomes joined to two other amino acids, forming a long, continuous, unbranched polymer called a polypeptide chain The amino acids that make up a polypeptide chain are joined by peptide bonds that result from the linkage of the carboxyl group of one amino acid to the amino group of its neighbor, with the elimination of a molecule of water (Figure 2.24c) A polypeptide chain composed of a string of amino acids joined by peptide bonds has the following backbone: R α − C H H + C O H N H O (a) Side Chain H R + N H α H C C H O Amino group − O Peptide bond Carboxyl group (b) R" R' N H N H C OH C H O + H N C C OH H H H O H2O R' H R" N C C N C C H H O H H O OH Peptide bond (c) FIGURE 2.24 Amino acid structure Ball-and-stick model (a) and chemical formula (b) of a generalized amino acid in which R can be any of a number of chemical groups (see Figure 2.26) (c) The formation of a peptide bond occurs by the condensation of two amino acids, drawn here in the uncharged state In the cell, this reaction occurs on a ribosome as an amino acid is transferred from a carrier (a tRNA molecule) onto the end of the growing polypeptide chain (see Figure 11.49) D -Alanine -Alanine L COOH C H Mirror CH3 NH2 CH3 NH2 COOH C H FIGURE 2.25 Amino acid stereoisomerism Because the ␣-carbon of all amino acids except glycine is bonded to four different groups, two stereoisomers can exist The D and L forms of alanine are shown O H C N H R C H C C N R O H O H C N H R C C C R O H The “average” polypeptide chain contains about 450 amino acids The longest known polypeptide, found in the muscle protein titin, contains more than 30,000 amino acids Once incorporated into a polypeptide chain, amino acids are termed residues The residue on one end of the chain, the N-terminus, contains an amino acid with a free (unbonded) ␣-amino group, whereas the residue at the opposite end, the C-terminus, has a free ␣-carboxyl group In addition to amino acids, many proteins contain other types of components that are added after the polypeptide is synthesized These include carbohydrates (to form glycoproteins), metal-containing groups (to form metalloproteins) and organic groups (e.g., flavoproteins) The Properties of the Side Chains The backbone, or main chain, of the polypeptide is composed of that part of each amino acid that is common to all of them The side chain or R group (Figure 2.24), bonded to the ␣-carbon, is highly variable among the 20 building blocks, and it is this variability that ultimately gives proteins their diverse structures and activities If the various amino acid side chains are considered together, they exhibit a large variety of structural features, ranging from fully charged to hydrophobic, and they can participate in a wide variety of covalent and noncovalent bonds As discussed in the following chapter, the side chains of the “active sites” of enzymes can facilitate (catalyze) many different organic reactions The assorted characteristics of the side chains of the amino acids are important in both intramolecular interactions, which determine the structure and activity of the molecule, and intermolecular interactions, which determine the relationship of a polypeptide with other molecules, including other polypeptides (page 59) Amino acids are classified on the character of their side chains They fall roughly into four categories: polar and charged, polar and uncharged, nonpolar, and those with unique properties (Figure 2.26) Polar, charged Amino acids of this group include aspartic acid, glutamic acid, lysine, and arginine These four amino acids contain side chains that become fully charged; that JWCL151_ch02_031-083.qxd 6/10/09 1:44 PM Page 51 + Polar charged NH3 C NH CH2 O– O – + NH3 NH C CH2 CH2 C CH2 CH2 CH2 CH2 CH2 CH2 O O + – + H3N C C O – H O Aspartic acid (Asp or D) – + H3N C C O H O Glutamic acid (Glu or E) CH2 CH2 + H3N C C O HC NH + CH C NH – + H3N C C O– H3N C C O H O Lysine (Lys or K) H O Arginine (Arg or R) H O Histidine (His or H) Properties of side chains (R groups): Hydrophilic side chains act as acids or bases which tend to be fully charged (+ or –) under physiologic conditions Side chains form ionic bonds and are often involved in chemical reactions Polar uncharged O C OH CH3 CH2 C – H3N C C O + H O Threonine (Thr or T) NH2 CH2 CH2 CH2 – H3N C C O + H O Serine (Ser or S) O CH2 H C OH – H3N C C O + OH NH2 + H O Glutamine (Gln or Q) – H3N C C O – H3N C C O + H O Asparagine (Asn or N) H O Tyrosine (Tyr or Y) Properties of side chains: Hydrophilic side chains tend to have partial + or – charge allowing them to participate in chemical reactions, form H-bonds, and associate with water Nonpolar CH3 CH3 CH3 CH3 + H3N C C O– CH3 CH + H3N C C O– H O Alanine (Ala or A) CH3 CH S CH2 CH2 H C CH3 CH2 + H3N C C O– H O Valine (Val or V) CH3 + H3N C C O– H O Leucine (Leu or L) H O Isoleucine (Ile or I) NH C CH CH2 + H3N C C O– H O Methionine (Met or M) CH2 + H3N C C O– CH2 + H3N C C O– H O Phenylalanine (Phe or F) H O Tryptophan (Trp or W) Properties of side chains: Hydrophobic side chain consists almost entirely of C and H atoms These amino acids tend to form the inner core of soluble proteins, buried away from the aqueous medium They play an important role in membranes by associating with the lipid bilayer Side chains with unique properties H + – H3N C C O H O Glycine (Gly or G) Side chain consists only of hydrogen atom and can fit into either a hydrophilic or hydrophobic environment.Glycine often resides at sites where two polypeptides come into close contact + SH CH2 CH2 CH2 CH2 CH C O– N O + H2 Proline (Pro or P) – H3N C C O H O Cysteine (Cys or C) Though side chain has polar, uncharged character, it has the unique property of forming a covalent bond with another cysteine to form a disulfide link FIGURE 2.26 The chemical structure of amino acids These 20 amino acids represent those most commonly found in proteins and, more specifically, those encoded by DNA Other amino acids occur as the result of a modification to one of those shown here The amino acids are Though side chain has hydrophobic character, it has the unique property of creating kinks in polypeptide chains and disrupting ordered secondary structure arranged into four groups based on the character of their side chains, as described in the text All molecules are depicted as free amino acids in their ionized state as they would exist in solution at neutral pH 51 JWCL151_ch02_031-083.qxd 52 6/10/09 1:44 PM Page 52 Chapter THE CHEMICAL BASIS OF LIFE is, the side chains contain relatively strong organic acids and bases The ionization reactions of glutamic acid and lysine are shown in Figure 2.27 At physiologic pH, the side chains of these amino acids are almost always present in the fully charged state Consequently, they are able to form ionic bonds with other charged species in the cell For example, the positively charged arginine residues of histone proteins are linked by ionic bonds to the negatively charged phosphate groups of DNA (see Figure 2.3) Histidine is also considered a polar, charged amino acid, though in most cases it is only partially charged at physiologic pH In fact, because of its ability to gain or lose a proton in physiologic pH ranges, histidine is a particularly important residue in the active site of many proteins (as in Figure 3.13) OH O CH2 CH2 – O O C C – CH2 + CH2 OH H N C C N C C H H O H H O + H + H + (a) H + NH2 NH2 CH2 CH2 CH2 – CH2 OH CH2 H + CH2 CH2 + CH2 N C C N C C H H O H H O Polar, uncharged The side chains of these amino acids have a partial negative or positive charge and thus can form hydrogen bonds with other molecules including water These amino acids are often quite reactive Included in this category are asparagine and glutamine (the amides of aspartic acid and glutamic acid), threonine, serine, and tyrosine Nonpolar The side chains of these amino acids are hydrophobic and are unable to form electrostatic bonds or interact with water The amino acids of this category are alanine, valine, leucine, isoleucine, tryptophan, phenylalanine, and methionine The side chains of the nonpolar amino acids generally lack oxygen and nitrogen They vary primarily in size and shape, which allows one or another of them to pack tightly into a particular space within the core of a protein, associating with one another as the result of van der Waals forces and hydrophobic interactions The other three amino acids—glycine, proline, and cysteine—have unique properties that separate them from the others The side chain of glycine consists of only a hydrogen atom, and glycine is a very important amino acid for just this reason Owing to its lack of a side chain, glycine residues provide a site where the backbones of two polypeptides (or two segments of the same polypeptide) can approach one another very closely In addition, glycine is more flexible than other amino acids and allows parts of the backbone to move or form a hinge Proline is unique in having its ␣-amino group as part of a ring (making it an imino acid) Proline is a hydrophobic amino acid that does not readily fit into an ordered secondary structure, such as an ␣ helix (page 54), often producing kinks or hinges Cysteine contains a reactive sulfhydryl (—SH) group and is often covalently linked to another cysteine residue, as a disulfide (—SS—) bridge (b) FIGURE 2.27 The ionization of charged, polar amino acids (a) The side chain of glutamic acid loses a proton when its carboxylic acid group ionizes The degree of ionization of the carboxyl group depends on the pH of the medium: the greater the hydrogen ion concentration (the lower the pH), the smaller the percentage of carboxyl groups that are present in the ionized state Conversely, a rise in pH leads to an increased ionization of the proton from the carboxyl group, increasing the percentage of negatively charged glutamic acid side chains The pH at which 50 percent of the side chains are ionized and 50 percent are unionized is called the pK, which is 4.4 for the side chain of free glutamic acid At physiologic pH, virtually all of the glutamic acid residues of a polypeptide are negatively charged (b) The side chain of lysine becomes ionized when its amino group gains a proton The greater the hydroxyl ion concentration (the higher the pH), the smaller the percentage of amino groups that are positively charged The pH at which 50 percent of the side chains of lysine are charged and 50 percent are uncharged is 10.0, which is the pK for the side chain of free lysine At physiologic pH, virtually all of the lysine residues of a polypeptide are positively charged Once incorporated into a polypeptide, the pK of a charged group can be greatly influenced by the surrounding environment Cysteine H H O H H O N C C N C C CH2 CH2 Oxidation SH SH S + 2H+ + 2e– S Reduction CH2 CH2 N C C N C C H H O H H O Disulfide bridges often form between two cysteines that are distant from one another in the polypeptide backbone or even in two separate polypeptides Disulfide bridges help stabilize the intricate shapes of proteins, particularly those present outside of cells where they are subjected to added physical and chemical stress JWCL151_ch02_031-083.qxd 6/10/09 1:44 PM Page 53 2.5 FOUR TYPES OF BIOLOGICAL MOLECULES Not all of the amino acids described in this section are found in all proteins, nor are the various amino acids distributed in an equivalent manner A number of other amino acids are also found in proteins, but they arise by alterations to the side chains of the 20 basic amino acids after their incorporation into a polypeptide chain For this reason they are called posttranslational modifications (PTMs) Dozens of different types of PTMs have been documented The most widespread and important PTM is the reversible addition of a phosphate group to a serine, threonine, or tyrosine residue PTMs can generate dramatic changes in the properties and function of a protein, most notably by modifying its threedimensional structure, level of activity, localization within the cell, life span, and/or its interactions with other molecules The presence or absence of a single phosphate group on a key regulatory protein has the potential to determine whether or not a cell will behave as a cancer cell or a normal cell Because of PTMs, a single polypeptide can exist as a number of distinct biological molecules The ionic, polar, or nonpolar character of amino acid side chains is very important in protein structure and function Most soluble (i.e., nonmembrane) proteins are constructed so that the polar residues are situated at the surface of the molecule where they can associate with the surrounding water and contribute to the protein’s solubility in aqueous solution (a) FIGURE 2.28 Disposition of hydrophilic and hydrophobic amino acid residues in the soluble protein cytochrome c (a) The hydrophilic side chains, which are shown in green, are located primarily at the surface of the protein where they contact the surrounding aqueous medium (b) The hydrophobic residues, which are shown in red, are located 53 (Figure 2.28a) In contrast, the nonpolar residues are situated predominantly in the core of the molecule (Figure 2.28b) The hydrophobic residues of the protein interior are often tightly packed together, creating a type of three-dimensional jigsaw puzzle in which water molecules are generally excluded Hydrophobic interactions among the nonpolar side chains of these residues are a driving force during protein folding (page 62) and contribute substantially to the overall stability of the protein In many enzymes, reactive polar groups project into the nonpolar interior, giving the protein its catalytic activity For example, a nonpolar environment can enhance ionic interactions between charged groups that would be lessened by competition with water in an aqueous environment Some reactions that might proceed at an imperceptibly slow rate in water can occur in millionths of a second within the protein The Structure of Proteins Nowhere in biology is the intimate relationship between form and function better illustrated than with proteins The structure of most proteins is completely defined and predictable Each amino acid in one of these giant macromolecules is located at a specific site within the structure, giving the protein the precise shape and reactivity required for the job at hand Protein structure can be described at several levels of organization, each emphasizing a (b) primarily within the center of the protein, particularly in the vicinity of the central heme group (ILLUSTRATION, IRVING GEIS IMAGE FROM IRVING GEIS COLLECTION/HOWARD HUGHES MEDICAL INSTITUTE RIGHTS OWNED BY HHMI REPRODUCED BY PERMISSION ONLY.) JWCL151_ch02_031-083.qxd 54 6/10/09 1:44 PM Page 54 Chapter THE CHEMICAL BASIS OF LIFE different aspect and each dependent on different types of interactions Customarily, four such levels are described: primary, secondary, tertiary, and quaternary The first, primary structure, concerns the amino acid sequence of a protein, whereas the latter three levels concern the organization of the molecule in space To understand the mechanism of action and biological function of a protein it is essential to know how that protein is constructed Primary Structure The primary structure of a polypeptide is the specific linear sequence of amino acids that constitute the chain With 20 different building blocks, the number of different polypeptides that can be formed is 20n, where n is the number of amino acids in the chain Because most polypeptides contain well over 100 amino acids, the variety of possible sequences is essentially unlimited The information for the precise order of amino acids in every protein that an organism can produce is encoded within the genome of that organism As we will see later, the amino acid sequence provides the information required to determine a protein’s threedimensional shape and thus its function The sequence of amino acids, therefore, is all-important, and changes that arise in the sequence as a result of genetic mutations in the DNA may not be readily tolerated The earliest and best-studied example of this relationship is the change in the amino acid sequence of hemoglobin that causes the disease sickle cell anemia This severe, inherited anemia results solely from a single change in amino acid sequence within the hemoglobin molecule: a nonpolar valine residue is present where a charged glutamic acid is normally located This change in hemoglobin structure can have a dramatic effect on the shape of red blood cells, converting them from disk-shaped cells to sickle-shaped cells (Figure 2.29), which tend to clog small blood vessels, causing pain and life-threatening crises Not all amino acid changes have such a dramatic effect, as evidenced by the differences in amino acid sequence in the same protein among related organisms The degree to which changes in the primary sequence are toler- FIGURE 2.29 Scanning electron micrograph of a red blood cell from a person with sickle cell anemia Compare with the micrograph of a normal red blood cell of Figure 4.32a (COURTESY OF J T THORNWAITE, B F CAMERON, AND R C LEIF.) ated depends on the degree to which the shape of the protein or the critical functional residues are disturbed The first amino acid sequence of a protein was determined by Frederick Sanger and co-workers at Cambridge University in the early 1950s Beef insulin was chosen for the study because of its availability and its small size—two polypeptide chains of 21 and 30 amino acids each The sequencing of insulin was a momentous feat in the newly emerging field of molecular biology It revealed that proteins, the most complex molecules in cells, have a definable substructure that is neither regular nor repeating, unlike those of polysaccharides Each particular polypeptide, whether insulin or some other species, has a precise sequence of amino acids that does not vary from one molecule to another With the advent of techniques for rapid DNA sequencing (see Section 18.15), the primary structure of a polypeptide can be deduced from the nucleotide sequence of the encoding gene In the past few years, the complete sequences of the genomes of hundreds of organisms, including humans, have been determined This information will eventually allow researchers to learn about every protein that an organism can manufacture However, translating information about primary sequence into knowledge of higher levels of protein structure remains a formidable challenge Secondary Structure All matter exists in space and therefore has a three-dimensional expression Proteins are formed by linkages among vast numbers of atoms; consequently their shape is complex The term conformation refers to the threedimensional arrangement of the atoms of a molecule, that is, to their spatial organization Secondary structure describes the conformation of portions of the polypeptide chain Early studies on secondary structure were carried out by Linus Pauling and Robert Corey of the California Institute of Technology By studying the structure of simple peptides consisting of a few amino acids linked together, Pauling and Corey concluded that polypeptide chains exist in preferred conformations that provide the maximum possible number of hydrogen bonds between neighboring amino acids Two conformations were proposed In one conformation, the backbone of the polypeptide assumed the form of a cylindrical, twisting spiral called the alpha (␣) helix (Figure 2.30a,b) The backbone lies on the inside of the helix, and the side chains project outward The helical structure is stabilized by hydrogen bonds between the atoms of one peptide bond and those situated just above and below it along the spiral (Figure 2.30c) The X-ray diffraction patterns of actual proteins produced during the 1950s bore out the existence of the ␣ helix, first in the protein keratin found in hair and later in various oxygen-binding proteins, such as myoglobin and hemoglobin (see Figure 2.34) Surfaces on opposite sides of an ␣ helix may have contrasting properties In water-soluble proteins, the outer surface of an ␣ helix often contains polar residues in contact with the solvent, whereas the surface facing inward typically contains nonpolar side chains The second conformation proposed by Pauling and Corey was the beta (␤)-pleated sheet, which consists of several segments of a polypeptide lying side by side Unlike the coiled, cylindrical form of the ␣ helix, the backbone of each segment of JWCL151_ch02_031-083.qxd 6/10/09 1:44 PM Page 55 2.5 FOUR TYPES OF BIOLOGICAL MOLECULES H C C N C CR O C N N C C C R N N C C C N C C C C N C C N C (b) N C C N H H O R C H C H N R C N C CH O N O H C R C H N O H C C R H N O H C R C N C O R C H O H R 3.6 residues C C O C N N (a) C H C O C O R NC H H C H N C O H C C N 55 H (c) FIGURE 2.30 The alpha helix (a) Linus Pauling (left) and Robert Corey with a wooden model of the alpha helix The model has a scale of inch per Å, which represents a 254 million-fold enlargement (b) The helical path around a central axis taken by the polypeptide backbone in a region of ␣ helix Each complete (360Њ) turn of the helix corresponds to 3.6 amino acid residues The distance along the axis between adjacent residues is 1.5 Å (c) The arrangement of the atoms of the backbone of the ␣ helix and the hydrogen bonds that form between amino acids Because of the helical rotation, the peptide bonds of every fourth amino acid come into close proximity The approach of the carbonyl group (CPO) of one peptide bond to the imine group (HON) of another peptide bond results in the formation of hydrogen bonds between them The hydrogen bonds (orange bars) are essentially parallel to the axis of the cylinder and thus hold the turns of the chain together (A: COURTESY polypeptide (or ␤ strand) in a ␤ sheet assumes a folded or pleated conformation (Figure 2.31a) Like the ␣ helix, the ␤ sheet is also characterized by a large number of hydrogen bonds, but these are oriented perpendicular to the long axis of the polypeptide chain and project across from one part of the chain to another (Figure 2.31b) Like the ␣ helix, the ␤ sheet has also been found in many different proteins Because ␤ strands are highly extended, the ␤ sheet resists pulling (tensile) forces Silk is composed of a protein containing an extensive amount of ␤ sheet; silk fibers are thought to owe their strength to this architectural feature Remarkably, a single fiber of spider silk, which may be a tenth the thickness of a human hair, is roughly five times stronger than a steel fiber of comparable weight Those portions of a polypeptide chain not organized into an ␣ helix or a ␤ sheet may consist of hinges, turns, loops, or fingerlike extensions Often, these are the most flexible portions of a C C N N R R R C C C C N C N C N N C C C N C C C R R R R o 7.0A ARCHIVES, CALIFORNIA INSTITUTE OF TECHNOLOGY.) R C (a) (b) C OF THE N FIGURE 2.31 The ␤-pleated sheet (a) Each polypeptide of a ␤ sheet assumes an extended but pleated conformation referred to as a ␤ strand The pleats result from the location of the ␣-carbons above and below the plane of the sheet Successive side chains (R groups in the figure) project upward and downward from the backbone The distance along the axis between adjacent residues is 3.5 Å (b) A ␤-pleated sheet consists of a number of ␤ strands that lie parallel to one another and are joined together by a regular array of hydrogen bonds between the carbonyl and imine groups of the neighboring backbones Neighboring segments of the polypeptide backbone may lie either parallel (in the same N-terminal S C-terminal direction) or antiparallel (in the opposite N-terminal S C-terminal direction) (ILLUSTRATION, IRVING GEIS IMAGE FROM THE IRVING GEIS COLLECTION/HOWARD HUGHES MEDICAL INSTITUTE RIGHTS OWNED BY HHMI REPRODUCTION BY PERMISSION ONLY.) JWCL151_ch02_031-083.qxd 56 6/10/09 1:44 PM Page 56 Chapter THE CHEMICAL BASIS OF LIFE FIGURE 2.33 An X-ray diffraction pattern of myoglobin The pattern FIGURE 2.32 A ribbon model of ribonuclease The regions of ␣ helix are depicted as spirals and ␤ strands as flattened ribbons with the arrows indicating the N-terminal S C-terminal direction of the polypeptide Those segments of the chain that not adopt a regular secondary structure (i.e., an ␣ helix or ␤ strand) consist largely of loops and turns and are shown in lime green Disulfide bonds are shown in blue (AFTER A DRAWING BY JANE S RICHARDSON.) polypeptide chain and the sites of greatest biological activity For example, antibody molecules are known for their specific interactions with other molecules (antigens); these interactions are mediated by a series of loops at one end of the antibody molecule (see Figures 17.15 and 17.16) The various types of secondary structures are most simply depicted as shown in Figure 2.32: ␣ helices are represented by helical ribbons, ␤ strands as flattened arrows, and connecting segments as thinner strands Tertiary Structure The next level above secondary structure is tertiary structure, which describes the conformation of the entire polypeptide Whereas secondary structure is stabilized primarily by hydrogen bonds between atoms that form the peptide bonds of the backbone, tertiary structure is stabilized by an array of noncovalent bonds between the diverse side chains of the protein Secondary structure is largely limited to a small number of conformations, but tertiary structure is virtually unlimited The detailed tertiary structure of a protein is usually determined using the technique of X-ray crystallography.5 of spots is produced as a beam of X-rays is diffracted by the atoms in the protein crystal, causing the X-rays to strike the film at specific sites Information derived from the position and intensity (darkness) of the spots can be used to calculate the positions of the atoms in the protein that diffracted the beam, leading to complex structures such as that shown in Figure 2.34 (COURTESY OF JOHN C KENDREW.) The three-dimensional structure of small proteins can also be determined by nuclear magnetic resonance (NMR) spectroscopy, which is not discussed in this text (see the supplement to the July issue of Nature Struct Biol., 1998, Nature Struct Biol 7:982, 2000, and Chem Rev 104:3517–3704, 2004 for reviews of this technology) Figure 1a, page 65, shows an NMR-derived structure In this technique (which is described in more detail in Sections 3.2 and 18.8), a crystal of the protein is bombarded by a thin beam of X-rays, and the radiation that is scattered (diffracted) by the electrons of the protein’s atoms is allowed to strike a radiation-sensitive plate or detector, forming an image of spots, such as those of Figure 2.33 When these diffraction patterns are subjected to complex mathematical analysis, an investigator can work backward to derive the structure responsible for producing the pattern It has become evident in recent years, as more and more protein structures have been solved, that a surprising number of proteins contain sizable segments that lack a defined conformation Examples of proteins containing these types of unstructured (or disordered ) segments can be seen in the models of the PrP protein in Figure on page 65 and the histone tails in Figure 12.9c The disordered regions in these proteins are depicted as dashed lines in the images, conveying the fact that these segments of the polypeptide can be present in many different positions and, thus, cannot be studied by X-ray crystallography Disordered segments tend to have a predictable amino acid composition, being enriched in charged and polar residues and deficient in hydrophobic residues You might be wondering whether proteins lacking a fully defined structure could be engaged in a useful function In fact, these disordered regions play key roles in vital cellular processes, often binding to DNA or to other proteins Remarkably, these segments JWCL151_ch02_031-083.qxd 6/10/09 1:44 PM Page 57 2.5 FOUR TYPES OF BIOLOGICAL MOLECULES often undergo a physical transformation once they bind to an appropriate partner and are then seen to possess a defined, folded structure Most proteins can be categorized on the basis of their overall conformation as being either fibrous proteins, which have an elongated shape, or globular proteins, which have a compact shape Most proteins that act as structural materials outside living cells are fibrous proteins, such as collagens and elastins of connective tissues, keratins of hair and skin, and silk These proteins resist pulling or shearing forces to which they are exposed In contrast, most proteins within the cell are globular proteins Myoglobin: The First Globular Protein Whose Tertiary Structure Was Determined The polypeptide chains of globular proteins are folded and twisted into complex shapes Distant points on the linear sequence of amino acids are brought next to each other and linked by various types of bonds The first glimpse at the tertiary structure of a globular protein came in 1957 through the X-ray crystallographic studies of John Kendrew and his colleagues at Cambridge University using X-ray diffraction patterns such as that shown in Figure 2.33 The protein they reported on was myoglobin Myoglobin functions in muscle tissue as a storage site for oxygen; the oxygen molecule is bound to an iron atom in the center of a heme group (The heme is an example of a prosthetic group, i.e., a portion of the protein that is not composed of amino acids, which is joined to the polypeptide chain after its assembly on the ribosome.) It is the heme group of myoglobin that gives most muscle tissue its reddish (a) FIGURE 2.34 The three-dimensional structure of myoglobin (a) The tertiary structure of whale myoglobin Most of the amino acids are part of ␣ helices The nonhelical regions occur primarily as turns, where the polypeptide chain changes direction The position of the heme is indicated in red (b) The three-dimensional structure of myoglobin (heme 57 color The first report on the structure of myoglobin provided a low-resolution profile sufficient to reveal that the molecule was compact (globular) and that the polypeptide chain was folded back on itself in a complex arrangement There was no evidence of regularity or symmetry within the molecule, such as that revealed in the earlier description of the DNA double helix This was not surprising considering the singular function of DNA and the diverse functions of protein molecules The earliest crude profile of myoglobin revealed eight rod-like stretches of ␣ helix ranging from to 24 amino acids in length Altogether, approximately 75 percent of the 153 amino acids in the polypeptide chain is in the ␣-helical conformation This is an unusually high percentage compared with that for other proteins that have since been examined No ␤-pleated sheet was found Subsequent analyses of myoglobin using additional X-ray diffraction data provided a much more detailed picture of the molecule (Figures 2.34a and 3.16) For example, it was shown that the heme group is situated within a pocket of hydrophobic side chains that promotes the binding of oxygen without the oxidation (loss of electrons) of the iron atom Myoglobin contains no disulfide bonds; the tertiary structure of the protein is held together exclusively by noncovalent interactions All of the noncovalent bonds thought to occur between side chains within proteins—hydrogen bonds, ionic bonds, and van der Waals forces—have been found (Figure 2.35) Unlike myoglobin, most globular proteins contain both ␣ helices and ␤ sheets Most importantly, these early landmark studies revealed that each protein has a unique tertiary structure that (b) indicated in red) The positions of all of the molecule’s atoms, other than hydrogen, are shown (A: ILLUSTRATION, IRVING GEIS IMAGE FROM IRVING GEIS COLLECTION/HOWARD HUGHES MEDICAL INSTITUTE RIGHTS OWNED BY HHMI REPRODUCED BY PERMISSION ONLY; B: KEN EWARD/PHOTO RESEARCHERS.) JWCL151_ch02_031-083.qxd 58 6/10/09 1:44 PM Page 58 Chapter THE CHEMICAL BASIS OF LIFE van der Waals forces troponin C bacterial phospholipase C CH CH3 CH3 CH3 CH3 CH3 CH3 CH Hydrogen bond C OH O C NH2 mamalian phospholipase C O CH2 CH2 CH2 CH2 – NH3+ O C CH2 Ionic bond FIGURE 2.35 Types of noncovalent bonds maintaining the conformation of proteins recoverin synaptotagmin can be correlated with its amino acid sequence and its biological function Protein Domains Unlike myoglobin, most eukaryotic proteins are composed of two or more spatially distinct modules, or domains, that fold independent of one another For example, the mammalian enzyme phospholipase C, shown in the central part of Figure 2.36, consists of four distinct domains, colored differently in the drawing The different domains of a polypeptide often represent parts that function in a semiindependent manner For example, they might bind different factors, such as a coenzyme and a substrate or a DNA strand and another protein, or they might move relatively independent of one another Protein domains are often identified with a specific function For example, proteins containing a PH domain bind to membranes containing a specific phospholipid, whereas proteins containing a chromodomain bind to a methylated lysine residue in another protein The functions of a newly identified protein can usually be predicted by the domains of which it is made Many polypeptides containing more than one domain are thought to have arisen during evolution by the fusion of genes that encoded different ancestral proteins, with each domain representing a part that was once a separate molecule Each domain of the mammalian phospholipase C molecule, for example, has been identified as a homologous unit in another protein (Figure 2.36) Some domains have been found only in one or a few proteins Other domains have been shuffled widely about during evolution, appearing in a variety of proteins whose other regions show little or no evidence of an evolutionary relationship Shuffling of domains creates pro- FIGURE 2.36 Proteins are built of structural units, or domains The mammalian enzyme phospholipase C is constructed of four domains, indicated in different colors The catalytic domain of the enzyme is shown in blue Each of the domains of this enzyme can be found independently in other proteins as indicated by the matching color (FROM LIISA HOLM AND CHRIS SANDER, STRUCTURE 5:167, 1997.) teins with unique combinations of activities On average, mammalian proteins tend to be larger and contain more domains than proteins of less complex organisms, such as fruit flies and yeast Dynamic Changes within Proteins Although X-ray crystallographic structures possess exquisite detail, they are static images frozen in time Proteins, in contrast, are not static and inflexible, but capable of considerable internal movements Proteins are, in other words, molecules with “moving parts.” Because they are tiny, nanometer-sized objects, proteins can be greatly influenced by the energy of their environment Random, small-scale fluctuations in the arrangement of the bonds within a protein create an incessant thermal motion within the molecule Spectroscopic techniques, such as nuclear magnetic resonance (NMR), can monitor dynamic movements within proteins, and they reveal shifts in hydrogen bonds, waving movements of external side chains, and the full rotation of the aromatic rings of tyrosine and phenylalanine residues about one of the single bonds The important role ... patch-clamp recording Cellular genes capable of causing malignant transformation Ability of RNA to catalyze reactions 19 91 1989 19 88 19 87 19 86 19 85 19 84 19 83 19 82 19 80 19 78 19 76 19 75 19 74 19 72 Rita Levi-Montalcini... Brief Contents 10 11 12 13 14 15 16 17 18 Introduction to the Study of Cell and Molecular Biology The Chemical Basis of Life Bioenergetics, Enzymes, and Metabolism 85 The Structure and Function... AMONG DIFFERENT SIGNALING PATHWAYS 638 18 Techniques in Cell and Molecular Biology 715 18 .1 THE LIGHT MICROSCOPE 716 Resolution 716 Visibility 717 Preparation of Specimens for Bright-Field Light